Differentiated HASTR/ci35 cells: A promising in vitro human astrocyte model for facilitating CNS drug development studies

Abstract

Astrocytes have shown longstanding promise as therapeutic targets for various central nervous system diseases. To facilitate drug development targeting astrocytes, we have recently developed a new conditionally immortalized human astrocyte cell line, termed HASTR/ci35 cells. In this study, in order to further increase their chances to contribute to various astrocyte studies, we report on the development of a culture method that improves HASTR/ci35 cell differentiation status and provide several proofs related to their astrocyte characteristics. The culture method is based on the simultaneous elimination of serum effects and immortalization signals. The results of qPCR showed that the culture method significantly enhanced several astrocyte marker gene expression levels. Using the differentiated HASTR/ci35, we examined their response profiles to nucleotide treatment and inflammatory stimuli, along with their membrane fatty acid composition. Consequently, we found that they responded to ADP or UTP treatment with a transient increase of intracellular Ca concentration, and that they could show reactive response to interleukin-1β treatments. Furthermore, the membrane phospholipids of the cells were enriched with polyunsaturated fatty acids. To summarize, as a unique human astrocyte model carrying the capability of a differentiation induction properties, HASTR/ci35 cells are expected to contribute substantially to astrocyte-oriented drug development studies.

Development, Characterization and Potential Applications of a Multicellular Spheroidal Human Blood–Brain Barrier Model Integrating Three Conditionally Immortalized Cell Lines

“…Using the hiMCS-BBB model, total RNA extraction and cDNA synthesis were prepared using the methods described previously. [23][24][25]29) Quantitative polymerase chain reaction (qPCR) was conducted using the cDNA and the primers listed in Table S2, and the amplification efficiency of each qPCR result was confirmed to be close to one. Data were obtained using the delta-delta-CT method, where the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level was used as a normalization control.…”

“…23) Immunocytochemistry (ICC) was then performed as described previously. [23][24][25]29) The primary and secondary antibodies used are listed in Table S1. All the antibodies were diluted to the indicated concentrations with CanGetSignal Immunostain Solution A (TOYOBO, Osaka, Japan).…”

“…Previously, we showed that the conditionally immortalized BBB cells undergo growth arrest and simultaneous differentiation upon elimination of their immortalization signal, which was achieved by changing the culture temperature from 33°C to 37°C (which causes tsSV40T protein degeneration). [23][24][25]29) Therefore, we tested the effects of 37°C culture temperature on spheroid formation. The results of 3D imaging analysis and hematoxylin and eosin staining showed that 37°C culture temperature suppressed spheroid growth (Figs.…”

Astrocyte Control of Zika Infection Is Independent of Interferon Type I and Type III Expression

“…There are differing opinions in the current scientific literature regarding the ability of astrocytes to control ZIKV infection [ 23 , 24 , 25 , 26 ]. Human astrocyte cell models are scarce, and in this study, we utilised a previously existing human astrocytic (astrocytoma) cell line (CCF-STTG1) [ 27 , 28 , 29 ] and two newly developed human immortalised primary astrocyte cell lines, foetal hTERT (abmGood, Richmond, BC, Canada) and foetal HASTRci35 [ 30 , 31 ]. Although these cell models were previously used in brain physiology and cancer-related research [ 31 , 32 , 33 , 34 , 35 , 36 ], their use in antiviral immunity is limited.…”

“…Human astrocyte cell models are scarce, and in this study, we utilised a previously existing human astrocytic (astrocytoma) cell line (CCF-STTG1) [ 27 , 28 , 29 ] and two newly developed human immortalised primary astrocyte cell lines, foetal hTERT (abmGood, Richmond, BC, Canada) and foetal HASTRci35 [ 30 , 31 ]. Although these cell models were previously used in brain physiology and cancer-related research [ 31 , 32 , 33 , 34 , 35 , 36 ], their use in antiviral immunity is limited. This work sought to analyse the antiviral response of multiple available human astrocyte cell models following Zika virus infection to understand the variability within astrocyte cell subsets in control ZIKV infection, as well as determine the ability of the pre-stimulation of the innate immune response to impact ZIKV infection in these model systems.…”